Nam On Ku

Apoptosis Generates Stable Fragments of Human Type I Keratins

Type I and II keratins help maintain the structural integrity of epithelial cells. Since apoptosis involves progressive cell breakdown, we examined its effect on human keratin polypeptides 8, 18, and 19 (K8, K18, K19) that are expressed in simple-type epithelia as noncovalent type I (K18, K19) and type II (K8) heteropolymers. Apoptosis induces rapid hyperphosphorylation of most known K8/18 phosphorylation sites and delayed formation of K18 and K19 stable fragments. In contrast, K8 is resistant to proteolysis and remains associated with the K18 fragments. Transfection of phosphorylation/glycosylation-mutant K8 and K18 does not alter fragment formation. The protein domains of the keratin fragments were determined using epitope-defined antibodies, and microsequencing indicated that K18 cleavage occurs at a conserved caspase-specific aspartic acid. The fragments are found preferentially within the detergent-insoluble pool and can be generated, in a phosphorylation-independent manner, by incubating keratins with caspase-3 or with detergent lysates of apoptotic cells but not with lysates of nonapoptotic cells. Our results indicate that type I keratins are targets of apoptosis-activated caspases, which is likely a general feature of keratins in most if not all epithelial cells undergoing apoptosis. Keratin hyperphosphorylation occurs early but does not render the keratins better substrates of the downstream caspases.

Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins

Members of the 14‐3‐3 protein family bind the human intermediate filament protein keratin 18 (K18) in vivo, in a cell‐cycle‐ and phosphorylation‐dependent manner. We identified K18 Ser33 as an interphase phosphorylation site, which increases its phosphorylation during mitosis in cultured cells and regenerating liver, and as an in vitro cdc2 kinase phosphorylation site. Comparison of wild‐type versus K18 Ser33→Ala/Asp transfected cells showed that K18 Ser33 phosphorylation is essential for the association of K18 with 14‐3‐3 proteins, and plays a role in keratin organization and distribution. Mutation of another K18 major phosphorylation site (Ser52) or K18 glycosylation sites had no effect on the binding of K18 to 14‐3‐3 proteins. The K18 phospho‐Ser33 motif is different from several 14‐3‐3‐binding phosphomotifs already described. Antibodies that are specific to K18 phospho‐Ser33 or phospho‐Ser52 show that although Ser52 and Ser33 phosphorylated K18 molecules manifest partial colocalization, these phosphorylation events reside predominantly on distinct K18 molecules. Our results demonstrate a unique K18 phosphorylation site that is necessary but not sufficient for K18 binding to 14‐3‐3 proteins. This binding is likely to involve one or more mitotic events coupled to K18 Ser33 phosphorylation, and plays a role in keratin subcellular distribution. Physiological Ser52 or Ser33 phosphorylation on distinct K18 molecules suggests functional compartmentalization of these modifications.

The cytoskeleton of digestive epithelia in health and disease

The mammalian cell cytoskeleton consists of a diverse group of fibrillar elements that play a pivotal role in mediating a number of digestive and nondigestive cell functions, including secretion, absorption, motility, mechanical integrity, and mitosis. The cytoskeleton of higher-eukaryotic cells consists of three highly abundant major protein families: microfilaments (MF), microtubules (MT), and intermediate filaments (IF), as well as a growing number of associated proteins. Within digestive epithelia, the prototype members of these three protein families are actins, tubulins, and keratins, respectively. This review highlights the important structural, regulatory, functional, and unique features of the three major cytoskeletal protein groups in digestive epithelia. The emerging exciting biological aspects of these protein groups are their involvement in cell signaling via direct or indirect interaction with a growing list of associated proteins (MF, MT, IF), the identification of several disease-causing mutations (IF, MF), the functional role that they play in protection from environmental stresses (IF), and their functional integration via several linker proteins that bridge two or potentially all three of these groups together. The use of agents that target specific cytoskeletal elements as therapeutic modalities for digestive diseases offers potential unique areas of intervention that remain to be fully explored.The mammalian cell cytoskeleton consists of a diverse group of fibrillar elements that play a pivotal role in mediating a number of digestive and nondigestive cell functions, including secretion, absorption, motility, mechanical integrity, and mitosis. The cytoskeleton of higher-eukaryotic cells consists of three highly abundant major protein families: microfilaments (MF), microtubules (MT), and intermediate filaments (IF), as well as a growing number of associated proteins. Within digestive epithelia, the prototype members of these three protein families are actins, tubulins, and keratins, respectively. This review highlights the important structural, regulatory, functional, and unique features of the three major cytoskeletal protein groups in digestive epithelia. The emerging exciting biological aspects of these protein groups are their involvement in cell signaling via direct or indirect interaction with a growing list of associated proteins (MF, MT, IF), the identification of several disease-causing mutations (IF, MF), the functional role that they play in protection from environmental stresses (IF), and their functional integration via several linker proteins that bridge two or potentially all three of these groups together. The use of agents that target specific cytoskeletal elements as therapeutic modalities for digestive diseases offers potential unique areas of intervention that remain to be fully explored.

Keratin 8 mutations in patients with cryptogenic liver disease.

Background About 10 percent of patients who undergo liver transplantation have cryptogenic liver disease. In animal models, the absence of heteropolymeric keratins 8 and 18 or the presence of mutant keratins in hepatocytes causes or promotes liver disease. We have previously described a mutation in the keratin 18 gene in a patient with cryptogenic cirrhosis, but the importance of mutations in the keratin 8 and keratin 18 genes in such patients is unclear. Methods We tested for mutations in the keratin 8 and keratin 18 genes in purified genomic DNA isolated from 150 explanted livers and 89 peripheral-blood specimens from three groups of patients: 55 patients with cryptogenic liver disease; 98 patients with noncryptogenic liver disease, with causes that included alcohol use, autoimmunity, drug use, and viral infections; and 86 randomly selected inpatients and outpatients who provided blood to the hematology laboratory. Results Of the 55 patients with cryptogenic liver disease, 3 had glycine-to-cysteine muta...

Keratin binding to 14-3-3 proteins modulates keratin filaments and hepatocyte mitotic progression

Keratin polypeptides 8 and 18 (K8/18) are the major intermediate filament proteins of simple-type epithelia. K18 Ser-33 phosphorylation regulates its binding to 14-3-3 proteins during mitosis. We studied the significance of keratin binding to 14-3-3 in transgenic mice that overexpress wild-type or Ser-33→Ala (S33A) K18. In S33A but not wild-type K18-overexpressing mice, pancreatic acinar cell keratin filaments retracted from the basal nuclear region and became apically concentrated. In contrast, K18 S33A had a minimal effect on hepatocyte keratin filament organization. Partial hepatectomy of K18-S33A-overexpressing mice did not affect liver regeneration but caused limited mitotic arrest, accumulation of abnormal mitotic figures, dramatic fragmentation of hepatocyte keratin filaments, with retention of a speckled 14-3-3ζ mitotic cell nuclear-staining pattern that usually becomes diffuse during mitosis. Hence, K18 Ser-33 phosphorylation regulates keratin filament organization in simple-type epithelia in vivo. Keratin binding to 14-3-3 may partially modulate hepatocyte mitotic progression, in association with nuclear redistribution of 14-3-3 proteins during mitosis.

A disease- and phosphorylation-related nonmechanical function for keratin 8

Keratin 8 (K8) variants predispose to human liver injury via poorly understood mechanisms. We generated transgenic mice that overexpress the human disease-associated K8 Gly61-to-Cys (G61C) variant and showed that G61C predisposes to liver injury and apoptosis and dramatically inhibits K8 phosphorylation at serine 73 (S73) via stress-activated kinases. This led us to generate mice that overexpress K8 S73-to-Ala (S73A), which mimicked the susceptibility of K8 G61C mice to injury, thereby providing a molecular link between K8 phosphorylation and disease-associated mutation. Upon apoptotic stimulation, G61C and S73A hepatocytes have persistent and increased nonkeratin proapoptotic substrate phosphorylation by stress-activated kinases, compared with wild-type hepatocytes, in association with an inability to phosphorylate K8 S73. Our findings provide the first direct link between patient-related human keratin variants and liver disease predisposition. The highly abundant cytoskeletal protein K8, and possibly other keratins with the conserved S73-containing phosphoepitope, can protect tissue from injury by serving as a phosphate “sponge” for stress-activated kinases and thereby provide a novel nonmechanical function for intermediate filament proteins.

Keratins let liver live: Mutations predispose to liver disease and crosslinking generates Mallory-Denk bodies.

Keratin polypeptides 8 and 18 (K8/K18) are the cytoskeletal intermediate filament proteins of hepatocytes while K8/K18/K19 are the keratins of hepatobiliary ductal cells. Hepatocyte K8/K18 are highly abundant and behave as stress proteins with injury‐inducible expression. Human association studies show that K8/K18 germline heterozygous mutations predispose to end‐stage liver disease of multiple etiologies (≈3 fold increased risk), and to liver disease progression in patients with chronic hepatitis C infection. These findings are supported by extensive transgenic mouse and ex vivo primary hepatocyte culture studies showing that K8 or K18 mutations predispose the liver to acute or subacute injury and promote apoptosis and fibrosis. Mutation‐associated predisposition to liver injury is likely related to mechanical and nonmechanical keratin functions including maintenance of cell integrity, protection from apoptosis and oxidative injury, serving as a phosphate sponge, regulation of mitochondrial organization/function and protein targeting. These functions are altered by mutation‐induced changes in keratin phosphorylation, solubility and filament organization/reorganization. Keratins are also the major constituents of Mallory‐Denk bodies (MDBs). A toxin‐induced K8>K18 ratio, and keratin crosslinking by transglutaminase‐2 play essential roles in MDB formation. Furthermore, intracellular or cell‐released K18 fragments, generated by caspase‐mediated proteolysis during apoptosis serve as markers of liver injury. Therefore, K8 and K18 are cytoprotective stress proteins that play a central role in guarding hepatocytes from apoptosis. Keratin involvement in liver disease is multi‐faceted and includes modulating disease progression upon mutation, formation of MDBs in response to unique forms of injury, and serving as markers of epithelial cell death. (HEPATOLOGY 2007;46:1639–1649.)

Susceptibility to hepatotoxicity in transgenic mice that express a dominant-negative human keratin 18 mutant.

Keratins 8 and 18 (K8/18) are intermediate filament phosphoglycoproteins that are expressed preferentially in simple-type epithelia. We recently described transgenic mice that express point-mutant human K18 (Ku, N.-O., S. Michie, R.G. Oshima, and M.B. Omary. 1995. J. Cell Biol. 131:1303-1314) and develop chronic hepatitis and hepatocyte fragility in association with hepatocyte keratin filament disruption. Here we show that mutant K18 expressing transgenic mice are highly susceptible to hepatotoxicity after acute administration of acetaminophen (400 mg/Kg) or chronic ingestion of griseofulvin (1.25% wt/wt of diet). The predisposition to hepatotoxicity results directly from the keratin mutation since nontransgenic or transgenic mice that express normal human K18 are more resistant. Hepatotoxicity was manifested by a significant difference in lethality, liver histopathology, and biochemical serum testing. Keratin glycosylation decreased in all griseofulvin-fed mice, whereas keratin phosphorylation increased dramatically preferentially in mice expressing normal K18. The phosphorylation increase in normal K18 after griseofulvin feeding appears to involve sites that are different to those that increase after partial hepatectomy. Our results indicate that hepatocyte intermediate filament disruption renders mice highly susceptible to hepatotoxicity, and raises the possibility that K18 mutations may predispose to drug hepatotoxicity. The dramatic phosphorylation increase in nonmutant keratins could provide survival advantage to hepatocytes.

Stress, Apoptosis, and Mitosis Induce Phosphorylation of Human Keratin 8 at Ser-73 in Tissues and Cultured Cells

Simple epithelia express keratins 8 (K8) and 18 (K18) as their major intermediate filament proteins. We previously showed that several types of cell stress such as heat and virus infection result in a distinct hyperphosphorylated form of K8 (termed HK8). To better characterize K8/18 phosphorylation, we generated monoclonal antibodies by immunizing mice with hyperphosphorylated keratins that were purified from colonic cultured human HT29 cells pretreated with okadaic acid. One antibody specifically recognized HK8, and the epitope was identified as 71LLpSPL which corresponds to K8 phosphorylation at Ser-73. Generation of HK8 occurs in mitotic HT29 cells, basal crypt mitotic cells in normal mouse intestine, and in regenerating mouse hepatocytes after partial hepatectomy. Prominent levels of HK8 were also generated in HT29 cells that were induced to undergo apoptosis using anisomycin or etoposide. In addition, mouse hepatotoxicity that is induced by chronic feeding with griseofulvin resulted in HK8 formation in the liver. Our results demonstrate that a “reverse immunological” approach, coupled with enhancing in vivo phosphorylation using phosphatase inhibitors, can result in the identification of physiologic phosphorylation states. As such, K8 Ser-73 phosphorylation generates a distinct HK8 species under a variety of in vivo conditions including mitosis, apoptosis, and cell stress. The low steady state levels of HK8 during mitosis, in contrast to stress and apoptosis, suggest that accumulation of HK8 may represent a physiologic stress marker for simple epithelia.

Mutation of human keratin 18 in association with cryptogenic cirrhosis.

Mutations in 11 of the more than 20 keratin intermediate filaments cause several epidermal and oral associated diseases. No disease-associated mutations have been described in keratin 8 or 18 (K8/18) which are the major keratin pair in simple-type epithelia, as found in the liver, pancreas, and intestine. However, transgenic mice that express mutant keratin 18 develop chronic hepatitis, and have an increased susceptibility to drug-induced hepatotoxicity. Also, ectopic expression of epidermal K14 in mouse liver results in chronic hepatitis, and disruption of mouse K8 leads to embryo lethality with extensive liver hemorrhage. We tested if patients with liver disease of unknown cause may harbor mutations in K18. We describe a his127-->leu (H127L) K18 mutation in a patient with cryptogenic cirrhosis that is germline transmitted. The K18 H127L isolated from the liver explant, or after expression in bacteria, showed an altered migration on two-dimensional gel analysis as compared with normal human liver or bacterially expressed K18. Electron microscopy of in vitro assembled K18 H127L and wild type K8 showed an assembly defect as compared with normal K8/18 assembly. Our results suggest that mutations in K18 may be predispose to, or result in cryptogenic cirrhosis in humans.